There is a fundamental gap in understanding whether the two nucleotide binding domains in ATP-binding cassette (ABC)- transporters are both catalytically active and how they orchestrate the action of ATP hydrolysis in converting chemical free energy into mechanical work. Filling this knowledge gap may generate important biomedical benefits by providing a molecular-level mechanism that aids designs of new therapeutic strategies in treating ABC-transporter related human diseases and clinical problems. Our long-term goal is to understand the general principles of how chemical catalysis and conformational dynamics are connected in ABC-transporters. The objective here is to determine the ATP hydrolysis mechanism in two specific members of ABC- transporters, by a new multiscale QM/MM simulation approach called Reaction Path Force Matching (RP-FM). Our central hypothesis is that the two active sites in NBDs of these systems function asymmetrically in hydrolyzing ATP, thereby allowing only one of them to access the catalytic competent configuration at a time. Regarding the detailed enzyme mechanism in NBDs, we hypothesized that two particular conserved residues, whose roles have remained elusive, participate in ATP hydrolysis explicitly and dynamically. Our hypotheses have been formulated based on biochemical studies, crystal structures, and our preliminary simulation results. The rationale for the proposed research is that, once the precise catalytic mechanism is determined for the single conformational state on the proposed systems, further experiments and simulations can be designed and performed to examine the catalytic activity as a function of multiple conformational states along the transporter cycle. We plan to test our central hypothesis by pursuing two Specific Aims: (1) extend and validate the RP-FM method for simulating complex systems; and (2) elucidate the precise ATP hydrolysis mechanism in the toxin transporter HlyB and the maltose transporter. Under the first aim, we will extend and validate the RP-FM approach for simulations of the well-characterized enzyme chorismate mutase system and methyl- triphosphate hydrolysis in aqueous solution. Under the second aim, RP-FM simulations will be employed to establish the ATPase mechanism in the two ABC-transporters and determine the extent to which the two actives sites in each of these systems are catalytically different. The proposed research is original and innovative because neither QM/MM free energy simulations, nor the RP-FM method, have been applied to study ATP hydrolysis mechanisms for any ABC-transporters; our exploratory work on the HlyB system represents the first study of this kind. Upon completion of this project, we expect that the RP-FM method will become available as a general tool for reliable simulations of enzyme mechanisms and a detailed description of ATP hydrolysis mechanism for the two ABC-transporters examined here will be obtained. Such information is extremely useful in understanding not only the two specific bacterial transporters, but also other members in the ABC-transporter family.